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J. Anat. (1991), 174, pp. 207-219 With 11 figures Printed in Great Britain
Innervation of hairs in the facial skin of marsupial mammals SAW-KIN LOO AND ZDENEK HALATA*
School of Anatomy, University of New South Wales, Sydney, Australia 2033 and *Abteilung fur Funktionelle Anatomie, Anatomisches Institut, University of Hamburg, Federal Republic of Germany
(Accepted 2 August 1990) INTRODUCTION
The innervation of hair follicles, especially those of vibrissae or sinus hairs, has been studied in placental mammals by many authors including Vincent (1913), Andres (1966) and Patrizi & Munger (1966). The innervation of guard and vellus hair follicles has also been studied in the monkey by Andres & von During (1973), Halata & Munger (1980) and Munger & Halata (1983). Lyne (1959) classified the living members of Marsupialia according to the distribution of vibrissae in the head and limbs but there have been few reports of hair innervation in these animals. Except for the study on the innervation of vibrissae hairs in the brush-tailed possum, Trichosurus vulpecula (Hollis & Lyne, 1974) there have been no reports on hair innervation in other Australian marsupial mammals and none at all on the innervation on guard and pelage hairs in these animals. In view of the unique zoological position occupied by marsupial mammals and the comparative rarity of many species, we were fortunate to obtain specimens from representatives of four Superfamilies of the Australian Metatheria to carry out a systematic study of the innervation of vibrissae, guard and pelage hair follicles. These were representatives of two Superfamilies in the Order Polyprotodonta and representatives of two Superfamilies in the Order Diprotodonta. Members of the Order Polyprotodonta were the long-nosed bandicoot, Perameles nasuta (Suborder Peralomorphia, Superfamily Perameloidea, Family Peramelidae) and the kowari, Dasyuroides byrnei (Suborder Dasyuromorphia, Superfamily Dasyuroidea, Family Dasyuridea). Members of the Order Diprotodonta were the brush-tailed possum, Trichosurus vulpecula (Superfamily Phalangeroidea, Family Phalangeridae) and the tammar wallaby, Macropus eugenii (Superfamily Macropodoidea, Family Macropodidae). In addition we also studied the innervation of these hairs in the American opossum Didelphis virginiana, which is a member of the Order Polyprotodonta, Suborder Didelphimorphia. The similarities or differences which exist between innervation of vibrissae and pelage hair follicles in the Metatheria and the Eutheria are at present not known. It is also not known if there are differences or similarities between the Australian Polyprotodonts and Diprotodonts and between the Australian and American representatives of the Order Polyprotodonta, with respect to hair follicle innervation. MATERIALS AND METHODS
Marsupial mammals used in this study were: two male bandicoots (Perameles nasuta), 4 kowaris (Dasyuroides byrnei), three brush-tailed possums (Trichosurus vulpecula), two tammar wallabies (Macropus eugenii) and 3 opossums (Didelphis
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virginiana). The bandicoots and brush-tailed possums were anaesthetised by intraperitoneal injections of sodium pentobarbitone (Nembutal) 50 mg per kg body weight and were perfused with Karnovsky's fixative, containing 3 % glutaraldehyde and 3 % paraformaldehyde in 0.1 M cacodylate buffer at pH 7-4. Tissues from the upper lip were further fixed for two hours in the same fixative, after which they were postfixed in 1 % osmium tetroxide at 4 °C for one hour. The blocks were then stained in a 2 % aqueous solution of uranyl acetate and lead citrate for one hour. They were dehydrated in alcohols followed by acetone and blocked in Durcupan Fluka. All the other animals were perfused with 6% glutaraldehyde in 0-2 M phosphate buffer. Tissues from the upper lip were further fixed in the same fixative for two hours, after which they were postfixed in 1% osmium tetroxide at 4°C for one hour, and embedded in Epon 812. These two different protocols were followed because the specimens were collected over a period of ten years. Semithin sections were stained with the techniques of Ito & Winchester (1963) or Laczko & Levai (1975). Thin sections were cut on a Reichert OM3 Ultratome with diamond knives. The sections were stained with lead citrate and uranyl acetate and viewed in a Philips 300 electron microscope. Material from Australian animals was obtained postmortem under Licence Nos. A16, FLF 811 and File No. AC 85/7 (Committee on the Use of Animals in Research and Teaching, University of New South Wales). OBSERVATIONS
Innervation of the skin between hair follicles Free nerve endings Free nerve endings were seen in the papillary layer of the dermis and in the connective tissue between hair follicles (Figs. 1, 2). They were of two types. The first type consisted of groups of fine nerve endings containing a few mitochondria, neurotubules and neurofilaments, enclosed together by a single Schwann cell lamella (Fig. 1). These nerve endings ranged from 0-2 ,um to 0-5 ,um in diameter. The other type had an average diameter of 0 5 ,tm - 1 ,um and contained clusters of mitochondria, vesicles, irregularly arranged neurotubules and neurofilaments and were incompletely surrounded by one Schwann cell lamella with many pinocytotic vesicles (Fig. 2). In several instances the nerve terminal came into contact with the basal lamina of the epidermis and occasionally penetrated the basal layer of the epidermis. In these areas the nerve terminals were surrounded partly by Schwann cells and partly by the basal lamina of the epidermis.
Innervation of pelage hairs Pelage hair follicles were approximately 35 ,m in diameter with hair fibres averaging 17 ,m in diameter. The external root sheaths of these hairs were about 2 cells thick. They were innervated by lanceolate nerve endings and free nerve endings. In transverse sections lanceolate terminals were arranged radially around the hair follicle. They were found in the connective tissue sheath around the hair follicle between the hair bulge and the sebaceous glands (Fig. 3). The hair bulge is at the region at the junction of the upper one-third and lower two-thirds of the hair follicle where the external root sheath of the hair is thickened. There were about 12-16 lanceolate terminals running longitudinally along each hair follicle. These nerve terminals were 0-8 ,um to 1 2 ,m in diameter. In hairs which were incompletely
Hair innervation in marsupial mammals
209
-2'~ Fig. 1. Electron micrograph of groups of nerve endings (02-{05 sm in diameter) enclosed by one Schwann cell lamella (arrow) in the papillary layer of the dermis. The epidermis is labelled (e). Kowari. x 8000. 1sm in diameter) filled with mitochondria and Fig. 2. Electron micrograph of free nerve endings (05i incompletely surrounded by one Schwann cell lamina with many pinocytotic vesicles (arrow) in the papillary layer of the dermis. The epidermis is labelled (e). Kowari. x 19000.
surrounded by sebaceous glands, lanceolate terminals occurred only in the areas where the sebaceous glands were present (Fig. 3). In cross-sections, the terminals were flattened and were covered by Schwann cells on their flattened sides, so that they appeared to be 'sandwiched' by Schwann cell lamellae which contained large numbers of pinocytotic vesicles (Fig. 4). Axonal spikes projected from these nerve terminals on to the basal lamina of the external root sheath of the hair follicle and into the connective tissue sheath of the hair follicle (Fig. 4). The nerve terminal contained clusters of mitochondria, vesicles, neurotubules and neurofilaments. External to these longitudinal lanceolate terminals, there were also transverse lanceolate terminals, which spiralled around the hair in an oblique to horizontal direction. These nerve terminals were supplied by the nerve fascicle which penetrated the glassy membrane of the hair in the lower third of the hair follicle. There were only two or three of these transverse nerve terminals to each hair follicle in contrast to the greater numbers of longitudinally-orientated lanceolate nerve terminals. These nerve terminals were 0-8,um to 12,um in diameter and were filled with mitochondria, vesicles, neurotubules and neurofilaments. Each was enclosed by one Schwann cell lamella (Fig. 4). Free nerve endings were present adjacent to lanceolate endings and were localised to the connective tissue, mainly in the angle between the hair follicle and its associated sebaceous glands. Merkel cells were not seen in pelage hairs. Innervation
of guard hairs
In the present study, specialised pelage hairs around the orifices such as the mouth and eyes are referred to as guard hairs. Guard hair follicles were approximately 50 ,um
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Fig. 3. Electron micrograph of cross-section of pelage hair showing lanceolate nerve terminals arranged around the hair follicle. Hairs are labelled (h), sebaceous glands (s), myelinated nerve bundles (n) and vascular spaces (v). Bandicoot. x 1000. Fig. 4. Higher magnification of part of pelage hair in Figure 3 showing lanceolate nerve terminals (1) sandwiched between Schwann cell lamellae (s). Arrows indicate the areas where the axonal spikes of lanceolate terminals contact the basal lamina of the hair follicle. Arrowheads indicate axonal spikes projecting into the surrounding connective tissue. Transverse lanceolate nerve terminals are labelled (t) and the hair (h). Bandicoot. x 8000.
Hair innervation in marsupial mammals
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1owan. x 6400.
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Fig 5 Electron micrograph of part of guard hair showing piiloRuffini nerve terminals (arrows) surrounded by Schwann cell lamellae with many pinocytotic vesicles. A septal-like cell and one of its processes are labelled (*), lanceolate terminal () and the exteral root sheath of the hair follcle (h). Kowari. x 6400.
in diameter with hair fibres ranging between 20 #sm to 25 ,tm in diameter. Their external root sheaths were 3-4 cells thick and connective tissue sheaths approximately 40 gsm thick. Vascular spaces were present external to the connective tissue sheaths around the middle third of the hair follicle. The hair follicles were innerva'ted by four types of nerve endings: free nerve endings, lanceolate nerve endings, pilo-Ruffini nerve endings and Merkel nerve endings.
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Free nerve endings and lanceolate nerve endings Morphologically the nerve endings were similar to those in pelage hairs, with the difference that longitudinal nerve endings were more numerous because of the larger diameter of the hair. As in pelage hairs, two or three transverse lanceolate nerve endings were seen in each hair.
Pilo-Ruffini nerve endings Pilo-Ruffini nerve terminals occurred in the angle between the hair follicle and its sebaceous glands. In longitudinal sections of the hair follicle, cross-sections of the circularly-orientated nerve terminals were arranged parallel to the long axis of the hair follicle. They were embedded in the specialised connective tissue of the hair follicle, where collagen fibrils were also arranged circularly around the hair follicle (Fig. 5). The nerve terminals ran parallel to the collagen fibrils and were incompletely surrounded by Schwann cell lamellae with many pinocytotic vesicles. Occasionally spike-like processes from these nerve terminals, covered only by the axolemma, projected into the surrounding connective tissue. These nerve terminals (1-2,m in diameter) contained mitochondria, vesicles, neurofilaments and neurotubules in irregular arrangements. The collagen fibrils were compartmentalised into bundles by cells with long processes (septal-like cells) (Fig. 5). Pilo-Ruffini terminals were supplied by myelinated nerve fibres with an average diameter of 2-4 /m. Merkel nerve endings Merkel cells, with their associated nerve endings, were found in that part of the hair follicle where the external root sheath was thick (Fig. 6). Lanceolate nerve terminals were also seen at this level. There were approximately 40-60 Merkel cells per hair. Merkel cells had flattened nuclei and were situated in the basal layer of the epidermal outer root sheath. Osmiophilic granules, 70-130 ,m in diameter, were concentrated in the basal portion of the cytoplasm where the associated nerve terminal was present. The Merkel cell membrane formed desmosomes with the cell membrane of surrounding keratinocytes, sent rod-shaped processes between the cells of the external root sheath and occasionally through the basal lamina of the external root sheath
(Fig. 6).
Nerve endings associated with Merkel cells contained mitochondria, vesicles, neurotubules and neurofilaments and, occasionally, glycogen granules. In rare instances they sent spike-like processes into the Merkel cell (Fig. 6). These nerve terminals were covered by Schwann cell lamellae and in several places only by the basal lamina of the external root sheath.
Innervation of vibrissae hairs Vibrissae hair follicles had a diameter of 650-700 ,m in the region where the external root sheath was thick. Hair fibres were about 350-400 ,um in diameter. They were surrounded by a blood sinus, which consisted of a ring sinus superiorly and a trabeculated sinus inferiorly. Free nerve endings, longitudinal lanceolate nerve terminals, two to three transverse lanceolate nerve terminals, Merkel nerve endings and lamellated corpuscles were seen.
Merkel nerve endings Merkel cells were seen in the basal layer of the external root sheath where it was thickened. There were about 800-1200 Merkel cells per hair follicle (Fig. 7). Merkel
Hair innervation in marsupial mammals
213
.: ...- .
V
Fig. 6. Electron micrograph of Merkel cell (m) in the basal layer of the external root sheath of guard hair. The nerve terminal is labelled (n) as are the rod-shaped processes of the Merkel cell (arrows). Spike-like process of a nerve terninal projecting into the Merkel cell is labelled (*). Bandicoot. x 8000.
cells and their associated nerve endings were similar in structure to those described for guard hairs. In the brush-tailed possum, synaptic-like thickenings of the cell membranes were found between the Merkel cell and its associated nerve ending and, in some cases, dense-cored granules of the Merkel cell were seen to fuse with the thickened membranes (Fig. 8).
Lanceolate nerve endings Lanceolate nerve terminals (1-3-5 #rm in diameter) were supplied by myelinated nerves (3-5,um in diameter) that penetrated the lower half of the hair follicle. They were present adjacent to the glassy membrane. As in guard hairs, they were present at the level where the Merkel cells were seen. Occasionally the myelinated nerve fibre supplying the longitudinal lanceolate nerve terminal was narrowed at the point where it lost its myelin sheath, after which the nerve terminal expanded in diameter (Fig. 10). Many mitochondria and neurofilaments in irregular arrangement were present in the nerve terminal. The nerve terminal itself was ensheathed by a single layer of Schwann cell lamella which had many pinocytotic vesicles on its cell membrane. As soon as it had lost its myelin sheath, the terminal portion of the lanceolate nerve ending sent out spike-like processes or axonal spurs in all directions. These processes were seen to push through spaces between lamellar cells, so that they came into direct contact with the glassy membrane of the hair follicle or the surrounding connective tissue (Fig. 10). Many spike-like processes were present and in some places they were only covered by
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Fig. 7. Electron micrograph of part of sinus hair showing Merkel cells (m) and their associated nerve endings (n). Lanceolate nerve terminals are labelled (l) and the lamellated corpuscle (c). Brush-tailed
possum. x 3200. Fig. 8. Part of Merkel cell (m) and its associated nerve ending (n). Synaptic-like region where the cell membranes are thickened is arrowed. Brush-tailed possum. x 40000. Fig. 9. Part of sinus hair (h) and lanceolate nerve terminal (1). Synaptic-like junctions between the nerve terminal and Schwann cell lamella are arrowed. Tammar wallaby. x 17000.
Hair innervation in marsupial mammals
215
"MAD AP,
A
.............
Fig. 10. Electron micrograph of part of sinus hair (h) with lanceolate terminal (0. (*) indicates area where the myelin sheath is lost. Spike-like processes are arrowed, Schwann cell lamellae are labelled (s) and the glassy membrane (g). Kowari. x 6400. Fig. 11. Higher magnification of lamellated corpuscle in Figure 7. The nerve terminal is labelled (n), Schwann cell lamellae (s) and an axonal spike is arrowed. Brush-tailed possum. x 8000.
Schwann cell basal lamina (Fig. 10). Thickenings of the plasma membrane resembling synaptic thickenings were seen between Schwann cell membrane and the axolemma of the lanceolate nerve terminal (Fig. 9). There were only a few transverse lanceolate terminals. They were surrounded by a single layer of Schwann cell lamellae with many
S.-K. LOO AND Z. HALATA 216 pinocytotic vesicles, or were sometimes surrounded by several layers of Schwann cell lamellae, resembling the simple corpuscles described below.
Lamellated corpuscles In the brush-tailed possum and the kowari, lamellated corpuscles were seen in the region of the hair follicle, where the transverse lanceolate nerve terminals were present. They consisted of a nerve terminal and 6-7 layers of lamellar cells in hemilamellar arrangement. The nerve terminal projected into the connective tissue between the radial clefts formed by the lamellar cells on either side of the corpuscle (Figs. 7, 11). There was no perineural capsule. In the kowari, the nerve terminal was arranged in a loose coil and was incompletely surrounded by lamellar cells. There was also no perineural capsule. In the tammar wallaby, simple lamellated capsules were seen in the connective tissue of the lower half of the hair follicle. In this animal some corpuscles were enveloped by one or two layers of Schwann cell lamellae with no perineural capsule, while others had a capsule of one to two layers of perineural cells. The nerve terminals in all cases were 1-5-3-5 ,um in diameter. They were supplied by myelinated nerves 2-4 ,um in diameter. Axonal spikes, without Schwann cell covering, projected from these endings into the surrounding connective tissue. The nerve endings were filled with mitochondria, a few vesicles and a few glycogen granules. DISCUSSION
The facial skin of the marsupial mammals investigated had all three types of hairs: pelage hairs, guard hairs and sinus or vibrissae hairs. We did not see hemi-sinus hairs of the type reported by Munger & Halata (1983) in primate facial skin. The pattern of innervation of the different types of hairs was similar in all species of animals examined in this investigation. Free nerve endings The two types of free nerve endings found resembled those described by Munger & Halata (1983); the nerve endings enclosed in groups by one Schwann cell lamella have been called 'penicillate nerve endings' by Cauna (1973).
Pelage hairs Pelage hairs were innervated by lanceolate nerve endings and free nerve endings. Lanceolate nerve terminals are classically described as nerve terminals ensheathed by Schwann cells, which run parallel to the long axis of the hair follicle (Andres, 1966). However, nerve terminals with a similar structure, which run more or less in a transverse direction, were consistently seen. They were situated just adjacent to the usual lanceolate nerve terminals. We have called them transverse lanceolate nerve terminals as opposed to the longitudinal lanceolate nerve terminals. They are different from the transverse nerve fibres running just below the level of the sebaceous glands in the sinus hairs of the rat (Bock, 1987). The transverse nerve fibres described by Bock were supplied by the superficial nerve plexus just below the epidermis, while the nerve endings described here, originated from myelinated nerve bundles from the lower third of the hair follicle. The longitudinal lanceolate terminals in this investigation were unusual in that they not only had spikes which projected on to the basal lamina of the external root sheath of the hair, but also many spikes which projected in the opposite direction externally into the surrounding connective tissue. Spikes of the type that
Hair innervation in marsupial mammals 217 projected into the surrounding connective tissue have so far only been described in relation to sinus hairs by Andres & von Diiring (1973), who speculated that they may play a role in the transduction process. From their morphology and location, they certainly seem to be eminently suitable for the detection of any movement in the hair or in the surrounding connective tissue. Furthermore, both types of lanceolate nerve terminals did not surround the whole hair follicle and were only present when sebaceous glands were present. This may indicate that the sebaceous glands and lanceolate terminals form a mechanoreceptor complex, as lanceolate terminals have been characterised by Iggo (1974) as rapidly adapting mechanoreceptors. On the other hand, the myelinated nerves which give rise to these terminals approach the hair follicle in the spaces between the sebaceous glands, so that the nerves would terminate further on and not in this region. Pilo-Ruffini nerve terminals as described by Biemesderfer, Munger, Binck & Dubner (1978) in primate facial skin, were not seen in pelage hairs in this investigation. Unlike guard hairs and sinus hairs which are considered to have a sensory function, pelage hairs are usually thought to be concerned with thermal insulation for maintenance of body heat in mammals. In the marsupial mammals investigated, pelage hairs are apparently sensitive structures as can be deduced from the pattern of their innervation. Axonal spikes found in these nerve terminals are also commonly found in free nerve endings (Munger & Halata, 1983), and in specialised sensory nerve endings like Pacinian and Meissner's corpuscles. We have also seen these spikes in Merkel cell nerve endings in the present investigation. Schoultz & Swett (1972) have postulated that increased tensile forces on collagen bundles could squeeze and distort axon terminals in Golgi-tendon organs constituting a mechanical force that could facilitate discharge in these axons. It is likely that any movement of the hair could cause the axonal spikes to be distorted so that they may play a role in the transduction process. Guard hairs Guard hairs in all the marsupial mammals investigated above, were very wellinnervated and had all the types of nerve endings described in vibrissae hairs of placental mammals (Andres, 1966; Patrizi & Munger, 1966; Yohro, 1977; Halata & Munger, 1980; Munger & Halata, 1983). In contrast to guard hairs in placental mammals and eyelashes in man (Munger & Halata, 1984), where only a few Merkel cells may be found, 40-60 Merkel cells and their associated nerve endings were regularly seen. These Merkel cells had the usual features described in the literature with the exception that, in the present study, we have seen spike-like processes that project from the associated nerve ending into the Merkel cell cytoplasm. As in vibrissae hairs, Merkel cells were seen where the external root sheath was thickest. This region has been called the Haarwulst by Horstmann (1957), and is the region where most of the nerve endings associated with the hair follicle, such as lanceolate nerve terminals and Merkel nerve endings, are found. The pilo-Ruffini nerve terminals were prominent and were situated in a more superficial position when compared with those in monkey sinus hairs and human eyelashes (Halata & Munger, 1980; Munger & Halata, 1984), where they occurred in the lower half of the hair follicle. It is likely from their innervation that guard hairs in these marsupials that we have studied have a similar function to vibrissae hairs in placental mammals.
ANA 174
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S.-K. LOO AND Z. HALATA
Vibrissae hairs Vibrissae hairs possessed most of the nerve endings described in guard hairs. We did not see pilo-Ruffini nerve endings, but corpuscular nerve endings were found at two different levels in different animals. In the brush-tailed possum and the kowari, these corpuscles were situated in the vicinity of the transverse lanceolate nerve endings. Therefore, it is likely that they are transverse lanceolate terminals that had acquired additional layers of Schwann cell covering. However, in the tammar wallaby, the corpuscles were found in the same position as in the cat vibrissae follicle (Halata, 1975), i.e. in the lower half of the hair follicle. They differed from those in the cat in that they appeared to be more primitive, because they had a simplified structure with only one to two layers of Schwann cell covering. Lanceolate nerve endings in vibrissae hairs showed an exceptionally large number of axonal spikes, which were in direct contact with the glassy membrane of the hair follicle or the surrounding connective tissue. This observation reinforces the idea that they may be involved with detection of mechanical distortion of the tissue surrounding the hair and hence the transduction process. The desmosome-like thickenings between the axolemma of the lanceolate nerve terminal and its surrounding Schwann cell is also a feature which has been observed in Pacinian corpuscles (personal observation). Membrane thickenings between the Merkel cell membrane and its nerve terminal, with dense-cored vesicles in the immediate vicinity, have been reported by several authors (Chen, Gerson & Meyer, 1973; Mihara, Hashimoto, Ueda & Kumakiri, 1979; Hartschuh & Weihe, 1980), who suggest that this appearance might imply that densecored vesicles may be secreted from the Merkel cell into the nerve terminal. In conclusion, lanceolate terminals were found in all the different types of hairs. Pelage hairs had many more lanceolate terminals than had been reported previously. Guard hairs in the marsupial mammals were particularly well-innervated with all types of nerve endings usually seen only in vibrissae hairs. Vibrissae hairs had the usual types of nerve endings with the exception that we did not find any pilo-Ruffini nerve terminals associated with these hairs in this study, and the lamellated corpuscles seen in vibrissae hairs had a simpler structure when compared with those in the cat and the monkey. It has been found from this study that the pattern of hair innervation is similar in representative members of several superfamilies in the marsupial mammals and also that the pattern resembles that found in placental mammals. As had been mentioned above, except for the study by Hollis & Lyne (1974) on the innervation of vibrissae hairs in the brush-tailed possum, this is the first study on hair innervation on representative members of four superfamilies in marsupial mammals. It is also the first report on the innervation of pelage and guard hairs in marsupial mammals. SUMMARY
The innervation of pelage, guard hairs and vibrissae hairs was studied in five species of marsupial mammals by means of electron microscopy for the first time. This study showed that members of different superfamilies in marsupial mammals displayed the same pattern of hair innervation. This also resembled the pattern seen in the placental mammals. All types of hairs had both longitudinal and transverse lanceolate nerve terminals. Pelage hairs did not have any Merkel cells. Guard hairs were very richly innervated and had free nerve endings, lanceolate nerve endings, many Merkel cells with their associated nerve endings and pilo-Ruffini nerve endings. Vibrissae hairs had
219 Hair innervation in marsupial mammals free nerve endings, Merkel nerve endings and lamellated corpuscles, but pilo-Ruffini nerve endings were not seen in this investigation. Because of the profusion and variety of innervation in guard hairs of the marsupial mammals, these hairs may have a similar function to vibrissae hairs in placental mammals.
We wish to thank Ms Jennifer Flux (University of New South Wales) and Mrs Tjandrawati C6llen, Ms Birgit Knutz and Mr Stefan Schillemeit (University of Hamburg) for excellent technical assistance. REFERENCES
ANDRES, K. H. (1966). Uber die Feinstruktur der Rezeptoren an Sinushaaren. Zeitschriftffur Zellforschung und mikroskopische Anatomie 75, 339-365. ANDRES, K. H. & VON DURING, M. (1973). Morphology of cutaneous receptors. In Handbook of Sensory Physiology (ed. H. Autrum, R. Jung, W. R. Loewenstein & D. M. MacKay), pp. 3-28. Berlin, New York: Springer. BIEMESDERFER, D., MUNGER, B. L., BINCK, J. & DuBNER, R. (1978). The pilo-Ruffini complex: A non-sinus hair and associated slowly-adapting mechanoreceptor in primate facial skin. Brain Research 142, 197-222. B6CK, P. (1987). Localization of mechanoreceptors in the vibrissae of the rat by staining for cholinesterase activity (ChE). Zeitschrift fur mikroskopisch-anatomische Forschung 101, 79-90. CAUNA, N. (1973). The free penicillate nerve endings of the human hairy skin. Journal of Anatomy 115, 227-288. CHEN, S. Y., GERSON, S. & MEYER, J. (1973). The fusion of Merkel cell granules with a synapse-like structure. Journal of Investigative Dermatology 61, 290-292. HALATA, Z. (1975). The mechanoreceptors of the mammalian skin. Ultrastructure and morphological classification. Advances in Anatomy and Embryology 50, 1-77. HALATA, Z. & MUNGER, B. L. (1980). Sensory nerve endings in Rhesus monkey sinus hairs. Journal of Comparative Neurology 192, 645-663. HARTSCHUH, W. & WEIHE, E. (1980). Fine structural analysis of the synaptic junction of Merkel cell-axoncomplexes. Journal of Investigative Dermatology 75, 159-165. HOLLIS, D. E. & LYNE, A. G. (1974). Innervation of vibrissae follicles in the marsupial Trichosurus vulpecula. Australian Journal of Zoology 22, 263-276. HORSTMANN, E. (1957). Die Haut. In Handbuch der mikroskopischen Anatomie des Menschen (ed. W. von Mollendorf), pp. 1-276. Berlin, Heidelberg: Springer. IGGo, A. (1974). Cutaneous receptors. In The Peripheral Nervous System, pp. 347-404. New York, London: Pergamon Press. ITO, S. & WINCHESTER, R. J. (1963). The fine structure of the gastric mucosa in the bat. Journal of Cell Biology 16, 541-578. LACZKO, J. & LEVAI, G. (1975). A simple differential staining method for semi-thin sections of ossifying cartilage and bone tissue embedded in epoxy resin. Mikroskopie 31, 1-4. LYNE, A. G. (1959). The systematic and adaptive significance of the vibrissae in the Marsupialia. Proceedings of the Zoological Society of London 133, 79-133. MIHARA, M., HASHIMOTO, K., UEDA, K. & KUMAKIRI, M. (1979). The specialized junctions between Merkel cell and neurite: an electron microscopic study. Journal of Investigative Dermatology 73, 325-334. MUNGER, B. L. & HALATA, Z. (1983). The sensory innervation of the primate facial skin. I. Hairy skin. Brain Research Reviews 5, 45-80. MUNGER, B. L. & HALATA, Z. (1984). The sensorineural apparatus of the human eyelid. American Journal of Anatomy 170, 181-204. PATRIZI, G. & MUNGER, B. L. (1966). The ultrastructure and innervation of rat vibrissae. Journal of Comparative Neurology 126, 423-436. SCHOULTZ, T. W. & SwErr, J. E. (1972). The fine structure of the Golgi tendon organs. Journal of Neurocytology 1, 1-26. VINCENT, S. B. (1913). The tactile hair of the white rat. Journal of Comparative Neurology 23, 1-36. YOHRO, T. (1977). Arrangement and structure of sinus hair muscles in the big-clawed shrew, Sorex unguiculatus. Journal of Morphology 153, 317-332.
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J. Anat. (1991), 174, pp. 207-219 With 11 figures Printed in Great Britain
Innervation of hairs in the facial skin of marsupial mammals SAW-KIN LOO AND ZDENEK HALATA*
School of Anatomy, University of New South Wales, Sydney, Australia 2033 and *Abteilung fur Funktionelle Anatomie, Anatomisches Institut, University of Hamburg, Federal Republic of Germany
(Accepted 2 August 1990) INTRODUCTION
The innervation of hair follicles, especially those of vibrissae or sinus hairs, has been studied in placental mammals by many authors including Vincent (1913), Andres (1966) and Patrizi & Munger (1966). The innervation of guard and vellus hair follicles has also been studied in the monkey by Andres & von During (1973), Halata & Munger (1980) and Munger & Halata (1983). Lyne (1959) classified the living members of Marsupialia according to the distribution of vibrissae in the head and limbs but there have been few reports of hair innervation in these animals. Except for the study on the innervation of vibrissae hairs in the brush-tailed possum, Trichosurus vulpecula (Hollis & Lyne, 1974) there have been no reports on hair innervation in other Australian marsupial mammals and none at all on the innervation on guard and pelage hairs in these animals. In view of the unique zoological position occupied by marsupial mammals and the comparative rarity of many species, we were fortunate to obtain specimens from representatives of four Superfamilies of the Australian Metatheria to carry out a systematic study of the innervation of vibrissae, guard and pelage hair follicles. These were representatives of two Superfamilies in the Order Polyprotodonta and representatives of two Superfamilies in the Order Diprotodonta. Members of the Order Polyprotodonta were the long-nosed bandicoot, Perameles nasuta (Suborder Peralomorphia, Superfamily Perameloidea, Family Peramelidae) and the kowari, Dasyuroides byrnei (Suborder Dasyuromorphia, Superfamily Dasyuroidea, Family Dasyuridea). Members of the Order Diprotodonta were the brush-tailed possum, Trichosurus vulpecula (Superfamily Phalangeroidea, Family Phalangeridae) and the tammar wallaby, Macropus eugenii (Superfamily Macropodoidea, Family Macropodidae). In addition we also studied the innervation of these hairs in the American opossum Didelphis virginiana, which is a member of the Order Polyprotodonta, Suborder Didelphimorphia. The similarities or differences which exist between innervation of vibrissae and pelage hair follicles in the Metatheria and the Eutheria are at present not known. It is also not known if there are differences or similarities between the Australian Polyprotodonts and Diprotodonts and between the Australian and American representatives of the Order Polyprotodonta, with respect to hair follicle innervation. MATERIALS AND METHODS
Marsupial mammals used in this study were: two male bandicoots (Perameles nasuta), 4 kowaris (Dasyuroides byrnei), three brush-tailed possums (Trichosurus vulpecula), two tammar wallabies (Macropus eugenii) and 3 opossums (Didelphis
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virginiana). The bandicoots and brush-tailed possums were anaesthetised by intraperitoneal injections of sodium pentobarbitone (Nembutal) 50 mg per kg body weight and were perfused with Karnovsky's fixative, containing 3 % glutaraldehyde and 3 % paraformaldehyde in 0.1 M cacodylate buffer at pH 7-4. Tissues from the upper lip were further fixed for two hours in the same fixative, after which they were postfixed in 1 % osmium tetroxide at 4 °C for one hour. The blocks were then stained in a 2 % aqueous solution of uranyl acetate and lead citrate for one hour. They were dehydrated in alcohols followed by acetone and blocked in Durcupan Fluka. All the other animals were perfused with 6% glutaraldehyde in 0-2 M phosphate buffer. Tissues from the upper lip were further fixed in the same fixative for two hours, after which they were postfixed in 1% osmium tetroxide at 4°C for one hour, and embedded in Epon 812. These two different protocols were followed because the specimens were collected over a period of ten years. Semithin sections were stained with the techniques of Ito & Winchester (1963) or Laczko & Levai (1975). Thin sections were cut on a Reichert OM3 Ultratome with diamond knives. The sections were stained with lead citrate and uranyl acetate and viewed in a Philips 300 electron microscope. Material from Australian animals was obtained postmortem under Licence Nos. A16, FLF 811 and File No. AC 85/7 (Committee on the Use of Animals in Research and Teaching, University of New South Wales). OBSERVATIONS
Innervation of the skin between hair follicles Free nerve endings Free nerve endings were seen in the papillary layer of the dermis and in the connective tissue between hair follicles (Figs. 1, 2). They were of two types. The first type consisted of groups of fine nerve endings containing a few mitochondria, neurotubules and neurofilaments, enclosed together by a single Schwann cell lamella (Fig. 1). These nerve endings ranged from 0-2 ,um to 0-5 ,um in diameter. The other type had an average diameter of 0 5 ,tm - 1 ,um and contained clusters of mitochondria, vesicles, irregularly arranged neurotubules and neurofilaments and were incompletely surrounded by one Schwann cell lamella with many pinocytotic vesicles (Fig. 2). In several instances the nerve terminal came into contact with the basal lamina of the epidermis and occasionally penetrated the basal layer of the epidermis. In these areas the nerve terminals were surrounded partly by Schwann cells and partly by the basal lamina of the epidermis.
Innervation of pelage hairs Pelage hair follicles were approximately 35 ,m in diameter with hair fibres averaging 17 ,m in diameter. The external root sheaths of these hairs were about 2 cells thick. They were innervated by lanceolate nerve endings and free nerve endings. In transverse sections lanceolate terminals were arranged radially around the hair follicle. They were found in the connective tissue sheath around the hair follicle between the hair bulge and the sebaceous glands (Fig. 3). The hair bulge is at the region at the junction of the upper one-third and lower two-thirds of the hair follicle where the external root sheath of the hair is thickened. There were about 12-16 lanceolate terminals running longitudinally along each hair follicle. These nerve terminals were 0-8 ,um to 1 2 ,m in diameter. In hairs which were incompletely
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-2'~ Fig. 1. Electron micrograph of groups of nerve endings (02-{05 sm in diameter) enclosed by one Schwann cell lamella (arrow) in the papillary layer of the dermis. The epidermis is labelled (e). Kowari. x 8000. 1sm in diameter) filled with mitochondria and Fig. 2. Electron micrograph of free nerve endings (05i incompletely surrounded by one Schwann cell lamina with many pinocytotic vesicles (arrow) in the papillary layer of the dermis. The epidermis is labelled (e). Kowari. x 19000.
surrounded by sebaceous glands, lanceolate terminals occurred only in the areas where the sebaceous glands were present (Fig. 3). In cross-sections, the terminals were flattened and were covered by Schwann cells on their flattened sides, so that they appeared to be 'sandwiched' by Schwann cell lamellae which contained large numbers of pinocytotic vesicles (Fig. 4). Axonal spikes projected from these nerve terminals on to the basal lamina of the external root sheath of the hair follicle and into the connective tissue sheath of the hair follicle (Fig. 4). The nerve terminal contained clusters of mitochondria, vesicles, neurotubules and neurofilaments. External to these longitudinal lanceolate terminals, there were also transverse lanceolate terminals, which spiralled around the hair in an oblique to horizontal direction. These nerve terminals were supplied by the nerve fascicle which penetrated the glassy membrane of the hair in the lower third of the hair follicle. There were only two or three of these transverse nerve terminals to each hair follicle in contrast to the greater numbers of longitudinally-orientated lanceolate nerve terminals. These nerve terminals were 0-8,um to 12,um in diameter and were filled with mitochondria, vesicles, neurotubules and neurofilaments. Each was enclosed by one Schwann cell lamella (Fig. 4). Free nerve endings were present adjacent to lanceolate endings and were localised to the connective tissue, mainly in the angle between the hair follicle and its associated sebaceous glands. Merkel cells were not seen in pelage hairs. Innervation
of guard hairs
In the present study, specialised pelage hairs around the orifices such as the mouth and eyes are referred to as guard hairs. Guard hair follicles were approximately 50 ,um
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Fig. 3. Electron micrograph of cross-section of pelage hair showing lanceolate nerve terminals arranged around the hair follicle. Hairs are labelled (h), sebaceous glands (s), myelinated nerve bundles (n) and vascular spaces (v). Bandicoot. x 1000. Fig. 4. Higher magnification of part of pelage hair in Figure 3 showing lanceolate nerve terminals (1) sandwiched between Schwann cell lamellae (s). Arrows indicate the areas where the axonal spikes of lanceolate terminals contact the basal lamina of the hair follicle. Arrowheads indicate axonal spikes projecting into the surrounding connective tissue. Transverse lanceolate nerve terminals are labelled (t) and the hair (h). Bandicoot. x 8000.
Hair innervation in marsupial mammals
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Fig 5 Electron micrograph of part of guard hair showing piiloRuffini nerve terminals (arrows) surrounded by Schwann cell lamellae with many pinocytotic vesicles. A septal-like cell and one of its processes are labelled (*), lanceolate terminal () and the exteral root sheath of the hair follcle (h). Kowari. x 6400.
in diameter with hair fibres ranging between 20 #sm to 25 ,tm in diameter. Their external root sheaths were 3-4 cells thick and connective tissue sheaths approximately 40 gsm thick. Vascular spaces were present external to the connective tissue sheaths around the middle third of the hair follicle. The hair follicles were innerva'ted by four types of nerve endings: free nerve endings, lanceolate nerve endings, pilo-Ruffini nerve endings and Merkel nerve endings.
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Free nerve endings and lanceolate nerve endings Morphologically the nerve endings were similar to those in pelage hairs, with the difference that longitudinal nerve endings were more numerous because of the larger diameter of the hair. As in pelage hairs, two or three transverse lanceolate nerve endings were seen in each hair.
Pilo-Ruffini nerve endings Pilo-Ruffini nerve terminals occurred in the angle between the hair follicle and its sebaceous glands. In longitudinal sections of the hair follicle, cross-sections of the circularly-orientated nerve terminals were arranged parallel to the long axis of the hair follicle. They were embedded in the specialised connective tissue of the hair follicle, where collagen fibrils were also arranged circularly around the hair follicle (Fig. 5). The nerve terminals ran parallel to the collagen fibrils and were incompletely surrounded by Schwann cell lamellae with many pinocytotic vesicles. Occasionally spike-like processes from these nerve terminals, covered only by the axolemma, projected into the surrounding connective tissue. These nerve terminals (1-2,m in diameter) contained mitochondria, vesicles, neurofilaments and neurotubules in irregular arrangements. The collagen fibrils were compartmentalised into bundles by cells with long processes (septal-like cells) (Fig. 5). Pilo-Ruffini terminals were supplied by myelinated nerve fibres with an average diameter of 2-4 /m. Merkel nerve endings Merkel cells, with their associated nerve endings, were found in that part of the hair follicle where the external root sheath was thick (Fig. 6). Lanceolate nerve terminals were also seen at this level. There were approximately 40-60 Merkel cells per hair. Merkel cells had flattened nuclei and were situated in the basal layer of the epidermal outer root sheath. Osmiophilic granules, 70-130 ,m in diameter, were concentrated in the basal portion of the cytoplasm where the associated nerve terminal was present. The Merkel cell membrane formed desmosomes with the cell membrane of surrounding keratinocytes, sent rod-shaped processes between the cells of the external root sheath and occasionally through the basal lamina of the external root sheath
(Fig. 6).
Nerve endings associated with Merkel cells contained mitochondria, vesicles, neurotubules and neurofilaments and, occasionally, glycogen granules. In rare instances they sent spike-like processes into the Merkel cell (Fig. 6). These nerve terminals were covered by Schwann cell lamellae and in several places only by the basal lamina of the external root sheath.
Innervation of vibrissae hairs Vibrissae hair follicles had a diameter of 650-700 ,m in the region where the external root sheath was thick. Hair fibres were about 350-400 ,um in diameter. They were surrounded by a blood sinus, which consisted of a ring sinus superiorly and a trabeculated sinus inferiorly. Free nerve endings, longitudinal lanceolate nerve terminals, two to three transverse lanceolate nerve terminals, Merkel nerve endings and lamellated corpuscles were seen.
Merkel nerve endings Merkel cells were seen in the basal layer of the external root sheath where it was thickened. There were about 800-1200 Merkel cells per hair follicle (Fig. 7). Merkel
Hair innervation in marsupial mammals
213
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Fig. 6. Electron micrograph of Merkel cell (m) in the basal layer of the external root sheath of guard hair. The nerve terminal is labelled (n) as are the rod-shaped processes of the Merkel cell (arrows). Spike-like process of a nerve terninal projecting into the Merkel cell is labelled (*). Bandicoot. x 8000.
cells and their associated nerve endings were similar in structure to those described for guard hairs. In the brush-tailed possum, synaptic-like thickenings of the cell membranes were found between the Merkel cell and its associated nerve ending and, in some cases, dense-cored granules of the Merkel cell were seen to fuse with the thickened membranes (Fig. 8).
Lanceolate nerve endings Lanceolate nerve terminals (1-3-5 #rm in diameter) were supplied by myelinated nerves (3-5,um in diameter) that penetrated the lower half of the hair follicle. They were present adjacent to the glassy membrane. As in guard hairs, they were present at the level where the Merkel cells were seen. Occasionally the myelinated nerve fibre supplying the longitudinal lanceolate nerve terminal was narrowed at the point where it lost its myelin sheath, after which the nerve terminal expanded in diameter (Fig. 10). Many mitochondria and neurofilaments in irregular arrangement were present in the nerve terminal. The nerve terminal itself was ensheathed by a single layer of Schwann cell lamella which had many pinocytotic vesicles on its cell membrane. As soon as it had lost its myelin sheath, the terminal portion of the lanceolate nerve ending sent out spike-like processes or axonal spurs in all directions. These processes were seen to push through spaces between lamellar cells, so that they came into direct contact with the glassy membrane of the hair follicle or the surrounding connective tissue (Fig. 10). Many spike-like processes were present and in some places they were only covered by
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Fig. 7. Electron micrograph of part of sinus hair showing Merkel cells (m) and their associated nerve endings (n). Lanceolate nerve terminals are labelled (l) and the lamellated corpuscle (c). Brush-tailed
possum. x 3200. Fig. 8. Part of Merkel cell (m) and its associated nerve ending (n). Synaptic-like region where the cell membranes are thickened is arrowed. Brush-tailed possum. x 40000. Fig. 9. Part of sinus hair (h) and lanceolate nerve terminal (1). Synaptic-like junctions between the nerve terminal and Schwann cell lamella are arrowed. Tammar wallaby. x 17000.
Hair innervation in marsupial mammals
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Fig. 10. Electron micrograph of part of sinus hair (h) with lanceolate terminal (0. (*) indicates area where the myelin sheath is lost. Spike-like processes are arrowed, Schwann cell lamellae are labelled (s) and the glassy membrane (g). Kowari. x 6400. Fig. 11. Higher magnification of lamellated corpuscle in Figure 7. The nerve terminal is labelled (n), Schwann cell lamellae (s) and an axonal spike is arrowed. Brush-tailed possum. x 8000.
Schwann cell basal lamina (Fig. 10). Thickenings of the plasma membrane resembling synaptic thickenings were seen between Schwann cell membrane and the axolemma of the lanceolate nerve terminal (Fig. 9). There were only a few transverse lanceolate terminals. They were surrounded by a single layer of Schwann cell lamellae with many
S.-K. LOO AND Z. HALATA 216 pinocytotic vesicles, or were sometimes surrounded by several layers of Schwann cell lamellae, resembling the simple corpuscles described below.
Lamellated corpuscles In the brush-tailed possum and the kowari, lamellated corpuscles were seen in the region of the hair follicle, where the transverse lanceolate nerve terminals were present. They consisted of a nerve terminal and 6-7 layers of lamellar cells in hemilamellar arrangement. The nerve terminal projected into the connective tissue between the radial clefts formed by the lamellar cells on either side of the corpuscle (Figs. 7, 11). There was no perineural capsule. In the kowari, the nerve terminal was arranged in a loose coil and was incompletely surrounded by lamellar cells. There was also no perineural capsule. In the tammar wallaby, simple lamellated capsules were seen in the connective tissue of the lower half of the hair follicle. In this animal some corpuscles were enveloped by one or two layers of Schwann cell lamellae with no perineural capsule, while others had a capsule of one to two layers of perineural cells. The nerve terminals in all cases were 1-5-3-5 ,um in diameter. They were supplied by myelinated nerves 2-4 ,um in diameter. Axonal spikes, without Schwann cell covering, projected from these endings into the surrounding connective tissue. The nerve endings were filled with mitochondria, a few vesicles and a few glycogen granules. DISCUSSION
The facial skin of the marsupial mammals investigated had all three types of hairs: pelage hairs, guard hairs and sinus or vibrissae hairs. We did not see hemi-sinus hairs of the type reported by Munger & Halata (1983) in primate facial skin. The pattern of innervation of the different types of hairs was similar in all species of animals examined in this investigation. Free nerve endings The two types of free nerve endings found resembled those described by Munger & Halata (1983); the nerve endings enclosed in groups by one Schwann cell lamella have been called 'penicillate nerve endings' by Cauna (1973).
Pelage hairs Pelage hairs were innervated by lanceolate nerve endings and free nerve endings. Lanceolate nerve terminals are classically described as nerve terminals ensheathed by Schwann cells, which run parallel to the long axis of the hair follicle (Andres, 1966). However, nerve terminals with a similar structure, which run more or less in a transverse direction, were consistently seen. They were situated just adjacent to the usual lanceolate nerve terminals. We have called them transverse lanceolate nerve terminals as opposed to the longitudinal lanceolate nerve terminals. They are different from the transverse nerve fibres running just below the level of the sebaceous glands in the sinus hairs of the rat (Bock, 1987). The transverse nerve fibres described by Bock were supplied by the superficial nerve plexus just below the epidermis, while the nerve endings described here, originated from myelinated nerve bundles from the lower third of the hair follicle. The longitudinal lanceolate terminals in this investigation were unusual in that they not only had spikes which projected on to the basal lamina of the external root sheath of the hair, but also many spikes which projected in the opposite direction externally into the surrounding connective tissue. Spikes of the type that
Hair innervation in marsupial mammals 217 projected into the surrounding connective tissue have so far only been described in relation to sinus hairs by Andres & von Diiring (1973), who speculated that they may play a role in the transduction process. From their morphology and location, they certainly seem to be eminently suitable for the detection of any movement in the hair or in the surrounding connective tissue. Furthermore, both types of lanceolate nerve terminals did not surround the whole hair follicle and were only present when sebaceous glands were present. This may indicate that the sebaceous glands and lanceolate terminals form a mechanoreceptor complex, as lanceolate terminals have been characterised by Iggo (1974) as rapidly adapting mechanoreceptors. On the other hand, the myelinated nerves which give rise to these terminals approach the hair follicle in the spaces between the sebaceous glands, so that the nerves would terminate further on and not in this region. Pilo-Ruffini nerve terminals as described by Biemesderfer, Munger, Binck & Dubner (1978) in primate facial skin, were not seen in pelage hairs in this investigation. Unlike guard hairs and sinus hairs which are considered to have a sensory function, pelage hairs are usually thought to be concerned with thermal insulation for maintenance of body heat in mammals. In the marsupial mammals investigated, pelage hairs are apparently sensitive structures as can be deduced from the pattern of their innervation. Axonal spikes found in these nerve terminals are also commonly found in free nerve endings (Munger & Halata, 1983), and in specialised sensory nerve endings like Pacinian and Meissner's corpuscles. We have also seen these spikes in Merkel cell nerve endings in the present investigation. Schoultz & Swett (1972) have postulated that increased tensile forces on collagen bundles could squeeze and distort axon terminals in Golgi-tendon organs constituting a mechanical force that could facilitate discharge in these axons. It is likely that any movement of the hair could cause the axonal spikes to be distorted so that they may play a role in the transduction process. Guard hairs Guard hairs in all the marsupial mammals investigated above, were very wellinnervated and had all the types of nerve endings described in vibrissae hairs of placental mammals (Andres, 1966; Patrizi & Munger, 1966; Yohro, 1977; Halata & Munger, 1980; Munger & Halata, 1983). In contrast to guard hairs in placental mammals and eyelashes in man (Munger & Halata, 1984), where only a few Merkel cells may be found, 40-60 Merkel cells and their associated nerve endings were regularly seen. These Merkel cells had the usual features described in the literature with the exception that, in the present study, we have seen spike-like processes that project from the associated nerve ending into the Merkel cell cytoplasm. As in vibrissae hairs, Merkel cells were seen where the external root sheath was thickest. This region has been called the Haarwulst by Horstmann (1957), and is the region where most of the nerve endings associated with the hair follicle, such as lanceolate nerve terminals and Merkel nerve endings, are found. The pilo-Ruffini nerve terminals were prominent and were situated in a more superficial position when compared with those in monkey sinus hairs and human eyelashes (Halata & Munger, 1980; Munger & Halata, 1984), where they occurred in the lower half of the hair follicle. It is likely from their innervation that guard hairs in these marsupials that we have studied have a similar function to vibrissae hairs in placental mammals.
ANA 174
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S.-K. LOO AND Z. HALATA
Vibrissae hairs Vibrissae hairs possessed most of the nerve endings described in guard hairs. We did not see pilo-Ruffini nerve endings, but corpuscular nerve endings were found at two different levels in different animals. In the brush-tailed possum and the kowari, these corpuscles were situated in the vicinity of the transverse lanceolate nerve endings. Therefore, it is likely that they are transverse lanceolate terminals that had acquired additional layers of Schwann cell covering. However, in the tammar wallaby, the corpuscles were found in the same position as in the cat vibrissae follicle (Halata, 1975), i.e. in the lower half of the hair follicle. They differed from those in the cat in that they appeared to be more primitive, because they had a simplified structure with only one to two layers of Schwann cell covering. Lanceolate nerve endings in vibrissae hairs showed an exceptionally large number of axonal spikes, which were in direct contact with the glassy membrane of the hair follicle or the surrounding connective tissue. This observation reinforces the idea that they may be involved with detection of mechanical distortion of the tissue surrounding the hair and hence the transduction process. The desmosome-like thickenings between the axolemma of the lanceolate nerve terminal and its surrounding Schwann cell is also a feature which has been observed in Pacinian corpuscles (personal observation). Membrane thickenings between the Merkel cell membrane and its nerve terminal, with dense-cored vesicles in the immediate vicinity, have been reported by several authors (Chen, Gerson & Meyer, 1973; Mihara, Hashimoto, Ueda & Kumakiri, 1979; Hartschuh & Weihe, 1980), who suggest that this appearance might imply that densecored vesicles may be secreted from the Merkel cell into the nerve terminal. In conclusion, lanceolate terminals were found in all the different types of hairs. Pelage hairs had many more lanceolate terminals than had been reported previously. Guard hairs in the marsupial mammals were particularly well-innervated with all types of nerve endings usually seen only in vibrissae hairs. Vibrissae hairs had the usual types of nerve endings with the exception that we did not find any pilo-Ruffini nerve terminals associated with these hairs in this study, and the lamellated corpuscles seen in vibrissae hairs had a simpler structure when compared with those in the cat and the monkey. It has been found from this study that the pattern of hair innervation is similar in representative members of several superfamilies in the marsupial mammals and also that the pattern resembles that found in placental mammals. As had been mentioned above, except for the study by Hollis & Lyne (1974) on the innervation of vibrissae hairs in the brush-tailed possum, this is the first study on hair innervation on representative members of four superfamilies in marsupial mammals. It is also the first report on the innervation of pelage and guard hairs in marsupial mammals. SUMMARY
The innervation of pelage, guard hairs and vibrissae hairs was studied in five species of marsupial mammals by means of electron microscopy for the first time. This study showed that members of different superfamilies in marsupial mammals displayed the same pattern of hair innervation. This also resembled the pattern seen in the placental mammals. All types of hairs had both longitudinal and transverse lanceolate nerve terminals. Pelage hairs did not have any Merkel cells. Guard hairs were very richly innervated and had free nerve endings, lanceolate nerve endings, many Merkel cells with their associated nerve endings and pilo-Ruffini nerve endings. Vibrissae hairs had
219 Hair innervation in marsupial mammals free nerve endings, Merkel nerve endings and lamellated corpuscles, but pilo-Ruffini nerve endings were not seen in this investigation. Because of the profusion and variety of innervation in guard hairs of the marsupial mammals, these hairs may have a similar function to vibrissae hairs in placental mammals.
We wish to thank Ms Jennifer Flux (University of New South Wales) and Mrs Tjandrawati C6llen, Ms Birgit Knutz and Mr Stefan Schillemeit (University of Hamburg) for excellent technical assistance. REFERENCES
ANDRES, K. H. (1966). Uber die Feinstruktur der Rezeptoren an Sinushaaren. Zeitschriftffur Zellforschung und mikroskopische Anatomie 75, 339-365. ANDRES, K. H. & VON DURING, M. (1973). Morphology of cutaneous receptors. In Handbook of Sensory Physiology (ed. H. Autrum, R. Jung, W. R. Loewenstein & D. M. MacKay), pp. 3-28. Berlin, New York: Springer. BIEMESDERFER, D., MUNGER, B. L., BINCK, J. & DuBNER, R. (1978). The pilo-Ruffini complex: A non-sinus hair and associated slowly-adapting mechanoreceptor in primate facial skin. Brain Research 142, 197-222. B6CK, P. (1987). Localization of mechanoreceptors in the vibrissae of the rat by staining for cholinesterase activity (ChE). Zeitschrift fur mikroskopisch-anatomische Forschung 101, 79-90. CAUNA, N. (1973). The free penicillate nerve endings of the human hairy skin. Journal of Anatomy 115, 227-288. CHEN, S. Y., GERSON, S. & MEYER, J. (1973). The fusion of Merkel cell granules with a synapse-like structure. Journal of Investigative Dermatology 61, 290-292. HALATA, Z. (1975). The mechanoreceptors of the mammalian skin. Ultrastructure and morphological classification. Advances in Anatomy and Embryology 50, 1-77. HALATA, Z. & MUNGER, B. L. (1980). Sensory nerve endings in Rhesus monkey sinus hairs. Journal of Comparative Neurology 192, 645-663. HARTSCHUH, W. & WEIHE, E. (1980). Fine structural analysis of the synaptic junction of Merkel cell-axoncomplexes. Journal of Investigative Dermatology 75, 159-165. HOLLIS, D. E. & LYNE, A. G. (1974). Innervation of vibrissae follicles in the marsupial Trichosurus vulpecula. Australian Journal of Zoology 22, 263-276. HORSTMANN, E. (1957). Die Haut. In Handbuch der mikroskopischen Anatomie des Menschen (ed. W. von Mollendorf), pp. 1-276. Berlin, Heidelberg: Springer. IGGo, A. (1974). Cutaneous receptors. In The Peripheral Nervous System, pp. 347-404. New York, London: Pergamon Press. ITO, S. & WINCHESTER, R. J. (1963). The fine structure of the gastric mucosa in the bat. Journal of Cell Biology 16, 541-578. LACZKO, J. & LEVAI, G. (1975). A simple differential staining method for semi-thin sections of ossifying cartilage and bone tissue embedded in epoxy resin. Mikroskopie 31, 1-4. LYNE, A. G. (1959). The systematic and adaptive significance of the vibrissae in the Marsupialia. Proceedings of the Zoological Society of London 133, 79-133. MIHARA, M., HASHIMOTO, K., UEDA, K. & KUMAKIRI, M. (1979). The specialized junctions between Merkel cell and neurite: an electron microscopic study. Journal of Investigative Dermatology 73, 325-334. MUNGER, B. L. & HALATA, Z. (1983). The sensory innervation of the primate facial skin. I. Hairy skin. Brain Research Reviews 5, 45-80. MUNGER, B. L. & HALATA, Z. (1984). The sensorineural apparatus of the human eyelid. American Journal of Anatomy 170, 181-204. PATRIZI, G. & MUNGER, B. L. (1966). The ultrastructure and innervation of rat vibrissae. Journal of Comparative Neurology 126, 423-436. SCHOULTZ, T. W. & SwErr, J. E. (1972). The fine structure of the Golgi tendon organs. Journal of Neurocytology 1, 1-26. VINCENT, S. B. (1913). The tactile hair of the white rat. Journal of Comparative Neurology 23, 1-36. YOHRO, T. (1977). Arrangement and structure of sinus hair muscles in the big-clawed shrew, Sorex unguiculatus. Journal of Morphology 153, 317-332.
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